Elevated postoperative carcinoembryonic antigen guides adjuvant chemotherapy for stage II colon cancer: a multicentre cohort retrospective study

Most clinical doctors rely on high-risk factors recommended by guidelines to decide whether to undergo adjuvant chemotherapy for stage II colon cancer. However, these high-risk factors do not include postoperative carcinoembryonic antigen (CEA). This study aims to explore the elevation of postoperative CEA as a risk factor, in addition to other high-risk factors, to guide adjuvant chemotherapy for patients with stage II colon cancer. A retrospective analysis was conducted on stage II colon cancer patients who underwent curative surgery at Yunnan Cancer Hospital and The Sixth Affiliated Hospital of Sun Yat-Sen University from April 2008 to January 2019. Patients were classified into three groups based on high-risk factors recommended by guidelines and postoperative CEA levels: low-risk with normal postoperative CEA, low-risk with elevated postoperative CEA and high-risk. COX regression analysis was used to identify independent prognostic factors affecting patients’ recurrence free survival (RFS). The Kaplan–Meier method was used to create the patients’ RFS curve. The restricted cubic spline (RCS) curve was used to assess the correlation between postoperative CEA and RFS on a continuous scale. Among 761 patients, there were 444 males (62.01%), with a median [IQR] age of 58.0 (18.0–88.0) years. A group of 425 high-risk patients had a 3-year RFS of 82.2% (95% CI 78.5–86.1%), while a group of 291 low-risk patients had a 3-year RFS of 89.7% (95% CI 86.1–93.5%). There was a statistically significant difference between the two groups (HR 1.83; 95% CI 1.22–2.74; P = 0.0067). Among them, the 3-year RFS of 261 low-risk patients with normal postoperative CEA was 93.6% (95% CI 90.5–96.8%), while the 3-year RFS of 30 low-risk patients with elevated postoperative CEA was 57.3% (95% CI 41.8–71.4%). There was a significant difference compared to the 3-year RFS of 425 high-risk patients (overall log-rank P < 0.0001). The multivariate analysis adjusted by the COX proportional hazards model showed that low-risk patients with elevated postoperative CEA patients (HR 14.95, 95% CI 4.51–49.63, P < 0.0001) was independently associated with a 3-year RFS. The restricted cubic spline model showed that in stage II colon cancer patients with tumor diameter > 1.955 ng/mL, the risk of postoperative recurrence increased with increasing postoperative CEA levels. Patients with elevated postoperative CEA levels have a significantly increased risk of recurrence. They should be included as high-risk factors to guide adjuvant chemotherapy for stage II colon cancer.

The Sixth Affiliated Hospital of Sun Yat-sen University uses the Alinity I immunoassay analyzer (Abbott Diagnostics, Chicago, USA) following the WHO standard method (code 73/601).The reference range for serum CEA is 0.0 to 5.0 ng/mL.Values above 5.0 ng/mL are considered elevated CEA, while values below 5.0 ng/mL are considered normal.
These risk factors include pT4, < 12 lymph node dissections, poor histological differentiation, bowel perforation or obstruction, lymphovascular invasion, neural invasion, positive CRM, mucinous carcinoma.The high-risk group is defined as patients who have one or multiple risk factors simultaneously.The low-risk group is defined as other patients who do not have any high-risk factors.

Exposures
Divide patients into three groups: the low-risk with normal postoperative CEA group, the low-risk with elevated postoperative CEA group, and the high-risk group.

Surveillance protocol
The clinical evaluation of the patient includes serum CEA level detection, physical examination, imaging examinations (CT/MRI/PET-CT), and colonoscopic biopsy.CEA levels should be measured every 3 to 6 months for a continuous period of 3 years.Imaging examinations, including plain and contrast-enhanced scans of the patient's chest, abdomen, and pelvis, should be performed at least once every 12 months or at least once every 3 years.Colonoscopy is performed once a year after surgery, and once every 3 years thereafter.Colonoscopy, histological examination or imaging examination confirms whether there is recurrence or distant metastasis in all cases.

Statistical analysis
Continuous variables were presented as mean ± standard deviation (SD) for normally distributed data or median (interquartile range) for skewed data.Categorical variables were presented as frequency or percentage.Chisquare or Fisher's exact test (for discrete variables) and unpaired t-test, Wilcoxon signed-rank test, or analysis of variance (ANOVA) for continuous variables were used to compare patient characteristics.Survival analysis was conducted using the Kaplan-Meier method and log-rank test.All P values below 0.05 were statistically significant.The COX proportional hazards regression model was used to evaluate factors independently associated with RFS.Variables included in the final multivariable model were selected based on their clinical relevance and statistical significance in univariate analysis (cutoff value, P < 0.05).Intestinal obstruction or perforation, and positive CRM were not included in the multivariable analysis due to their low positivity rates.The correlation between postoperative CEA and RFS was evaluated on a continuous scale using restricted cubic splines (RCS) curves.Subgroup analysis was performed based on known risk factors, and interaction tests were conducted through the COX regression model.The internal validation of the final multivariate model for RFS was performed through a bootstrap sampling procedure (n = 1000 samples) on a population with an overall recurrence risk score.Statistical analysis was conducted using R software (version 3.6.3;http:// www.R-proje ct.org), SPSS 28.0, and GraphPad Prism 8 for plotting 13 .

Results
A total of 2054 patients with stage I to III rectal cancer who underwent surgical resection at Yunnan Cancer Hospital and Sixth Affiliated Hospital of Sun Yat-sen University from 2008 to 2019 were retrospectively collected, and 1338 patients were excluded (see Fig. 1 for inclusion and exclusion criteria).Finally, 716 patients with stage II colorectal cancer were included.Patients were divided into 2 groups according to guideline-recommended risk factors (3-6), of which 291 were low-risk patients and 425 were high-risk patients.Of the 291 low-risk patients, 261 had normal postoperative CEA and 30 had elevated postoperative CEA (Fig. 1).Of the 761 patients, 444 (62.01%) were male, and the median [IQR] age was 58.0 (18.0-88.0)years.The follow-up time exceeded 3 years, and they met the inclusion criteria.There were 97 cases of local recurrence and distant metastases, with a recurrence rate of 12.75%.The median follow-up time was 49.73 (95% CI 45.73-51.10)months.The clinicopathological characteristics are shown in Table 1.
The 3-year RFS was 93.6% (95% CI 90.5-96.8%)for 261 low-risk patients with normal postoperative CEA, and 57.3% (95% CI 41.8-71.4%)for 30 low-risk patients with elevated postoperative CEA, showing a statistically significant difference compared to the 3-year RFS of 425 high-risk patients (overall log-rank P < 0.0001) (Fig. 2C).In both the chemotherapy group (Fig. 3C) and the non-chemotherapy group (Fig. 3F), there was a statistically significant difference among the three groups of patients, leading to similar results.

Multivariate analyses of all variables
Table 2 shows the univariate and multivariate analysis of factors related to RFS.In the univariate analysis, neural invasion, preoperative elevation of CEA and CA199, preoperative NLR ≥ 3, postoperative elevation of CEA and CA199, and postoperative NLR ≥ 3 were associated with shortened RFS (P < 0.05).Multivariate analysis showed that postoperative elevations of CEA (HR 4.79, 95% CI 2.65-8.65,P < 0.0001) and CA199 (HR 2.69, 95% CI 1.18-6.13,P = 0.0189) were independently associated with shorter RFS.After adjusting for confounding factors and incorporating multiple COX models, postoperative elevation of CEA in low-risk patients (HR, 14.95; 95% CI 4.51-49.63;P < 0.0001) was independently associated with 3-year RFS (Table 3).A restricted cubic spline model showed that the risk of recurrence after surgery increased with increasing postoperative CEA levels in stage II colon cancer patients with tumor diameter > 1.955 ng/mL (Fig. 4).Subgroup analysis of RFS also found  that postoperative elevation of CEA was independently associated with RFS, without interaction with other known clinicopathological factors related to prognosis (Fig. 5).

Discussion
There are limitations to identifying risk factors for guiding adjuvant chemotherapy in stage II colon cancer patients.This study suggests incorporating postoperative CEA levels as a risk factor to assess the risk of recurrence and guide chemotherapy.In multivariate analysis, postoperative elevation of CEA was identified as an independent prognostic parameter that may affect treatment decisions even in the absence of other risk factors.This study incorporated potential risk factors for colon cancer recurrence into a COX proportional hazards model and identified two independent risk factors: postoperative CEA and postoperative CA199.It is worth noting that preoperative elevation of CEA is not an independent risk factor for 3-year disease-free survival in stage II colon cancer patients, which is consistent with previous studies 12 .Using only the TNM staging system for prognostic stratification of colon cancer has some limitations.The internationally recognized serum CEA is an important prognostic indicator for colorectal cancer 14 .Postoperative CEA is an independent risk factor for 3-year recurrence-free survival in stage II colon cancer patients.A study 15 found that postoperative positive CEA and CEA increment were independent prognostic factors for stage II colon cancer.Patients with elevated postoperative CEA levels and positive CEA increments had the worst PFS and OS compared to other groups.
The results of this study can provide reference for adjuvant therapy in stage II rectal cancer after radical surgery.Prognostic factors are not only related to pathological staging (T4 and/or N2), but also to preoperative high CEA levels.The combination of pT, pN, and preoperative high CEA levels may be predictive factors for resistance to CapeOX adjuvant chemotherapy 16 .According to a study 17   Vol:.(1234567890) www.nature.com/scientificreports/Numerous previous studies have reported risk factors for postoperative recurrence in stage II colon cancer patients, but no positive results were found in this study except for elevated postoperative levels of CEA and CA199.Preoperative NLR was correlated with RFS and OS, indicating that NLR can be used as a tool to determine which patients should receive/avoid adjuvant chemotherapy, especially for left-sided colon cancer.Based on receiver operating characteristic (ROC) curve analysis, the cutoff value of NLR was 3 20 .There are studies  be paid during follow-up.Mucinous histology may be an indicator for improving survival in stage II colon cancer chemotherapy 24 .There is also evidence that there is no significant difference in tumor-specific survival between adenocarcinoma and signet ring cell carcinoma.Stage II signet ring cell carcinoma should not receive adjuvant chemotherapy 25 .The overall survival (OS) of stage II colon cancer with less than 8 cleared lymph nodes is poor 26 .Studies recommend clearing 20 or more lymph nodes for accurate postoperative staging 27 .Adjuvant chemotherapy should be considered during the treatment of stage III colon cancer patients aged 70 or above, but chemotherapy has limited efficacy for stage II colon cancer in elderly patients 28 .Given the increasing incidence of colon cancer in young patients, doctors are more aggressive in treating stage II colon cancer.However, evidence for this treatment is limited 29 , and over-treatment leading to treatment-related harm should be avoided.
The OS of patients with stage II colon cancer who underwent laparoscopic radical surgery is superior to those who underwent open radical surgery, especially for patients aged 75 or older 30 .In the largest group of stage II colon cancer patients evaluated so far 31 , regardless of treatment regimen, patient age, or high-risk pathological features, OS improvement is associated with adjuvant chemotherapy.The toxicity of the 3-month group was significantly lower than that of the 6-month group in chemotherapy cycle studies.Both 3-month CAPOX and 6-month FOLFOX can be used to treat stage II colorectal cancer patients 32 .The TOSCA trial confirmed that there was no significant difference in OS between the two groups.Compared with 5-FU/LV, FOLFOX is unlikely to be cost-effective 33 .Recent research 34 has shown that a 3-month CAPOX regimen can be an effective treatment option.The convenience, reduced toxicity, and cost of using CAPOX as an adjuvant for 3 months suggest it as a potential option for high-risk stage II colon cancer 35 .However, adjuvant chemotherapy did not significantly improve cancer-specific survival in patients with adverse features of stage II colon cancer.Other markers are needed to select appropriate patients for adjuvant therapy 36 .Two important risk factors, mismatch repair (MMR) gene expression and tumor budding (TB), were not included in this study.Previous studies did not find dMMR to have prognostic value in terms of overall and disease-free survival in patients with stage II colon cancer.The recurrence rate in patients with dMMR tumors was significantly reduced 37 .The survival rate of stage II dMMR colon cancer patients with high-risk factors is similar to that of patients without high-risk factors, regardless of the presence of KRAS mutations 38 .This study suggests that tumors with a pathological indicator of TB ≥ 5 may exhibit a high risk of recurrence and poor prognosis.The evaluation of TB may help identify patients suitable for neoadjuvant therapy 39 .The TB grading based on the ITBCC2016 criteria should be routinely evaluated in pathological practice and may improve the benefit of adjuvant chemotherapy for stage II colon cancer 40 .
There are limitations to this exploratory study.Firstly, due to its retrospective design, there were differences in the timing of postoperative CEA measurements.However, we selected values that were closest to the time of surgery.Patients who received adjuvant treatment beyond 12 weeks or received adjuvant treatment during the trial were excluded.Secondly, the limitations of this retrospective study include the lack of incorporation of mismatch repair gene status and tumor budding, which are important indicators.However, in the ESMO 5 and CSCO 3 guidelines, the population with high microsatellite instability caused by mismatch repair gene deficiency is small, and we prioritize T4 stage over high microsatellite instability.This study will continue to include more cases and wait for subsequent results to be published.

Conclusions
Patients with elevated postoperative CEA levels have a significantly increased risk of recurrence.Although the proportion of patients with postoperative CEA elevation and no high-risk factors is low, they should still be considered as high-risk factors to guide adjuvant chemotherapy after surgery for stage II colon cancer.

Figure 2 .
Figure 2. Kaplan-Meier curves of recurrence-free survival based on different grouping methods.(A) Postoperative normal CEA vs postoperative elevated CEA in the overall patient population.(B) High-risk vs low-risk in the overall patient population.(C) low-risk patients with postoperative normal CEA vs low-risk patients with postoperative elevated CEA vs high-risk in the overall patient population.(D) Postoperative normal CEA vs postoperative elevated CEA in low-risk patients.(E) Postoperative normal CEA vs postoperative elevated CEA in high-risk patients.(F) Adjuvant chemotherapy with postoperative elevated CEA vs Nonadjuvant chemotherapy.

Figure 3 .
Figure 3. Kaplan-Meier survival curves of recurrence-free survival rates based on different grouping methods in chemotherapy and non-chemotherapy groups.(A) Postoperative normal CEA vs postoperative elevated CEA in the adjuvant chemotherapy population.(B) High-risk vs low-risk in the adjuvant chemotherapy population.(C) low-risk patients with postoperative normal CEA vs low-risk patients with postoperative elevated CEA vs high-risk in the adjuvant chemotherapy population.(D) Postoperative normal CEA vs postoperative elevated CEA in the non-adjuvant chemotherapy.(E) High-risk vs low-risk in the non-adjuvant chemotherapy population.(F) low-risk patients with postoperative normal CEA vs low-risk patients with postoperative elevated CEA vs high-risk in the non-adjuvant chemotherapy population.

Figure 4 .
Figure 4.The relationship between postoperative CEA as a continuous variable and hazard ratio for recurrence.The red solid line represents the unadjusted hazard ratio, and the red dashed line represents the 95% confidence interval obtained from restricted cubic spline regression.